Research to develop instrumentatIon and methods for the sequence analysis of peptide antigens presented to the immune system in association with class I and class II molecules of the major histocompatibility complex is proposed. This is a necessary first step in the development of synthetic/recombinant vaccines or other modulators of the immune system that are effective against bacterial and viral infections, cancer, autoimmune disorders and tissue transplant rejection. Of highest priority is the development of methods for characterizing specific disease state associated antigens found in complex mixtures of several thousand self peptides. Multistage chromatography in conjunction with both microcapillary high performance liquid chromatography/ and high performance capillary electrophoresis/ electrospray ionization tandem mass spectrometry will be employed for this purpose. Both of the above systems will be coupled directly to cytotoxicity assays. Specific goals include the following: (a) to further improve the sensitivity of the tandem mass spectrometry method so as to facilitate sequence analysis of class I peptides at the low femtomole level, (b) to develop proteolytic digestion methods that will facilitate sequence analysis of class II antigens at the low femtomole level, (c) to sequence peptides presented by class I HLA- A2.l molecules on human melanoma cells that are recognized by melanoma specific cytotoxic T lymphocytes, (d) to sequence peptides presented by the human class I molecules, HLA-B7, Aw68, and Aw69, (e) to determine the structural basis of peptide interaction with class I, MHC molecules and to define peptide epitopes recognized by xenoreactive, HLA-A2.1 restricted CTL, (f) to sequence peptides processed from HIV virus proteins and presented in association the class 1, HLA-A2.l molecules, (g) to identify the range of endogenous influenza hemagglutinin pep tides associated with the murine MHC class 1 Kd molecule produced by the wild type hemagglutinin gene and synthetic minigenes and to identify endogenous hemagglutinin peptides that bind Kd but are not immunogenic.